Watching biology in action in billionths of a second

March 20, 1998

For the first time, scientists using extremely high powered x-rays and a special pulsed laser have succeeded in making a "movie" of a protein molecule at work carrying out a biological reaction under normal physiological conditions. The landmark finding, reported in today's issue of Science, helps solve a decades-old riddle of how a well-studied biomolecule works, and ushers in a new era in the study of molecular structure and function that may lead to development of useful new drugs.

The findings are the result of a million-fold improvement in time resolution of X-ray measurements that can record changes in the shape of the working protein that occur in billionths of a second. The experiments were performed by an international team at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France using an X-ray beam thousands of times more brilliant than any used previously.

"To see things moving very fast, I need a very fast shutter speed on my camera, and I need a very bright light to illuminate my subject," explains Keith Moffat, professor of biochemistry and molecular biology and director of the Consortium for Advanced Radiation Sources at the University of Chicago, who led the research team. "The very bright light is this brilliant X-ray beam, and we built a very fast X-ray shutter."

Moffat and colleagues from the University of Chicago, ESRF and the Institute of Physical and Chemical Research in Saitama, Japan, focused on a protein called myoglobin, which binds and stores oxygen in muscle, much as its close relative hemoglobin does in the blood. Myoglobin was the first protein molecule whose static structure—essentially a still picture taken with X-rays—was determined. It has posed a tantalizing riddle ever since.

Proteins, which are made as linear strings of amino acids, must ball up into complex and highly specific shapes to carry out their myriad functions as transporters, catalysts, hormones and receptors. When Sir John Kendrew determined the precise shape of myoglobin in 1960, it immediately posed a problem.

"Looking at the molecule, there was no obvious way for oxygen to get in and out," Moffat said. "People immediately realized the atoms must not be 'bolted down,' but are moving around, so channels open and close to allow oxygen to go in and out." Where are these channels? "The theoretical chemists have had a field day with that for 30 years," he said.

To obtain structures of myoglobin molecules in motion, Moffat and colleagues beamed X-rays at crystals of myoglobin molecules that had bound not oxygen, but carbon monoxide, which myoglobin only releases when it is bombarded by light. A special pulsed laser synchronized with the X-ray beam initiated the release of the carbon monoxide.

What does myoglobin look like in action? The new results confirm many of the theoretical computations, but revise the picture somewhat, Moffat said. "Now we really see the tunnel opening," he said.

This "time-resolved" crystallography is the type of experiment that will soon be performed at the Advanced Photon Source at Argonne National Laboratory in Illinois. Moffat expects the even more brilliant X-rays at that synchrotron to improve the time resolution by another factor of 10.

Seeing biomolecules in their true dynamic state would be a boon to drug developers.

"There's a tremendous push to understand the structure of molecules and how they interact with drugs," Moffat said. "But that interaction is not a static event. If we understand not only what the enzyme looks like, but also how it wiggles and jiggles as it goes about its business, chemists may be able to design better drugs. Don't forget, if I just showed you a static picture of myoglobin, you'd tell me oxygen couldn't even get in and out."

Other authors are Vukica Srajer, Tsu-yi Teng, Zhong Ren and Wilfried Schildkamp from the University of Chicago; Thomas Ursby, Dominique Bourgeois and Michael Wulff from ESRF; and Shin-ichi Adachi from the Institute of Physical and Chemical Research in Japan.

The research was funded by the National Institutes of Health and was initiated by a grant from the Keck Foundation.

Summary:For the first time, scientists have succeeded in making a "movie" of a protein molecule at work under normal conditions. The landmark finding, reported in Science, ushers in a new era in the study of molecular structure and function that may lead to development of new drugs.